a. Mechanism of Inhibition
The class of enzymes known as amino acid decarboxylases (DC's) is of great interest from both a mechanistic and a therapeutic point of view. Mechanistically, this enzymatic family presents a curious dichotomy. Nature has chosen two very different cofactors to facilitate cleavage of the C.sub..alpha.- CO.sub.2 bond. Pyridoxal phosphate is used by the vast majority of amino acid decarboxylases studied to date. However, some enzymes, such as S-adenosylmethionine decarboxylase (SAM DC), use pyruvamide to achieve the same task. It is generally accepted that both cofactors form Schiff bases with the .alpha.-amino group of substrate and act as electron sinks, providing for delocalization of the negative charge in the nascent .alpha.-carbanion formed upon enzymatic decarboxylation. Overall, each catalyzes the net loss of CO.sub.2 from, and addition of a proton to, the .alpha.-carbon. With the exception of meso-diaminopimelate decarboxylase, all amino acid decarboxylases studied perform this sequence with retention of configuration. It is also noteworthy that while a few irreversible inhibitors have been developed for pyruvamide dependent decarboxylases (e.g. SAM DC), these inhibitors lack carboxyl groups. Decarboxylation-dependent irreversible inhibitors for pyruvamide-dependent decarboxylases have not yet been developed.
In seeking to develop irreversible, enzyme-activated inhibitors, the general strategy has been to divert the nascent .alpha.-carbanion from .alpha.-protonation (i.e. turnover) in one of two ways: (1) by placing a leaving group (typically fluoride or chloride) at the .beta.-carbon (.alpha.-halogenmethyl amino acids); or (2) by placing a .pi.-system adjacent to the carbanion (.alpha.-vinyl, .alpha.-ethynyl and .alpha.-allenyl amino acids). The most important members of these inhibitor classes are collected in Table 1 below.
TABLE 1 __________________________________________________________________________ Known Mechanism-Based Inhibitors of Amino Acid Decarboxylases Enzyme Enzyme Comm. No. Inhibitor(s) Potential Application __________________________________________________________________________ L-DOPA Decarboxylase E.C. 4.1.1.28 antihypertensive, Parkinson 's disease (DOPA comb. therapy) - R = vinyl, ethynyl, allenyl, CF.sub.2 H, CFH.sub.2, CHFCl(m-tyr.) - L-Ornithine Decarboxylase E.C. 4.1.1.17 antineoplastic, treatment of trypanosomiasis and pneumonia - R = vinyl, ethynyl, CF.sub.2 H, CFH.sub.2, CHFCl S-Adenosyl- Methionine Decarboxylase (Pyruvamide) E.C. 4.1.1.50 antineoplastic, treatment of trypanosomiasis - L-Lysine Decarboxylase E.C. 4.1.1.18 antimycoplasmic - L-Histidine Decarboxylase (PLP, pyruvamide) E.C. 4.1.1.22 antihistamine, antineoplastic - L-Arginine Decarboxylase E.C. 4.1.1.19 antibiotic, antiproliferative - R = CFH.sub.2, CF.sub.2 H L-Glutamate Decarboxylase E.C. 4.1.1.15 investigative tool; reduction of brain GABA levels - R = vinyl, CHFCl __________________________________________________________________________
.alpha.-Allenic-DOPA is a more effective inhibitor (shorter t-1/2, more complete inactivation) than either .alpha.-vinyl- or .alpha.-ethynyl-DOPA, although all three inactivate DOPA DC. This result shows that substitution of a three-carbon unit in place of the .alpha.-proton is quite tolerable, at least for DOPA DC.
The stereospecificity of inhibition has been studied for several of the .alpha.-halogenmethyl amino acids and is highly variable. For .alpha.-fluoromethyl DOPA, only the expected (S)-antipode inhibits DOPA DC. But for .alpha.-fluoromethylhistamine the unexpected (S)-antipode is the more potent inhibitor of histidine DC. For .alpha.-chlorofluoromethylornithine, all four possible stereoisomers irreversibly inhibit ornithine DC, all with t-1/2's between 2 and 4 minutes. The enantiospecificity of inhibition of the .alpha.-vinyl amino acids has yet to be determined.
The prior art discloses only three labeling studies involving irreversible inhibitors for amino acid decarboxylases in which the chemical nature of the covalent enzyme-inhibitor or cofactor-inhibitor adduct has been investigated. Glutamate decarboxylase inactivation by L-serine O-sulfate represents the original case in which Metzler put forth his enamine mechanism. (Likos, J. J.; Ueno, H.; Feldhaus, R. W.; Metzler, D. E. "A Novel Reaction of the Coenzyme of Glutamate Decarboxylase with L-Serine O-Sulfate," Biochemstry 1984, 23:5188-5194). However, this inhibitor is not stringently mechanism-based as it functions via enzyme-catalyzed .beta.-elimination rather than decarboxylation. On the other hand, .alpha.-fluoromethylhistidine is a good example of a mechanism-based decarboxylase inhibitor that also apparently follows a Metzler enamine mechanism (See Scheme 1 shown below). (Hayashi, H.; Tanase, S.; Snell, E. E. "Pyridoxal 5'-Phosphate-dependent Histidine Decarboxylase; Inactivation by .alpha.-Fluoromethylhistidine," J. Biol. Chem. 1986, 261, 11003-11009). ##STR8##
The postulated mechanism of action of .alpha.-fluoromethylhistidine involves enzyme-catalyzed decarboxylation followed by .beta.-fluoride-elimination. Transaldimination with an enzymatic lysine residue apparently ensues, leading to release of enamine 4. Nucleophilic attack of this enamine upon C-4 of the lysylpyridoximine results in the formation of a ternary enzyme-cofactor-inhibitor adduct 5. However, 5 can break down via a retro-Michael addition to release the enzymatic lysine residue and give a binary cofactor-inhibitor adduct 6. In certain cases binary adducts of this type (or the corresponding ketones) are released from the enzyme. In this case, the binary adduct is apparently intercepted via Michael addition of serine-322 in the wild-type enzyme. However, Snell's group has shown that the corresponding Ser.fwdarw.Ala322 mutant is also essentially irreversibly inactivated by .alpha.-fluoromethylhistidine. They propose avid noncovalent binding of the pyridoxylidene imine 6 (or the corresponding ketone) to the enzyme to account for this result. Other (non-decarboxylating) PLP-linked enzymes, for which evidence in support of a Metzler enamine mechanism has been obtained, include aspartate aminotransferase (serine O-sulfate), ornithine aminotransferase (4-aminohex-5-ynoate), .gamma.-aminobutyrate transaminase (4-amino-5-fluoropentanoate and 4-aminohex-5-enoate), and alanine racemase (.beta.-fluoroalanine, .beta.-chloroalanine and O-acetylserine).
In the case of ornithine decarboxylase inhibition by difluoromethylornithine (DFMO), labeling studies support a Michael addition-elimination pathway (See Scheme 2 shown below) (Poulin, R.; Lu, L.; Ackermann, B.; Bey, P.; Pegg, A. E., "Mechanism of the Irreversible Inactivation of Mouse Ornithine Decarboxylase by .alpha.-Difluoromethylornithine." J. Biol Chem. 1992, 265, 150-158). The trapped enzyme nucleophile in this case is a cysteine residue. A related Michael addition-elimination, in which lysine-38 is the enzyme nucleophile, appears to be operative in the inhibition of alanine racemase by .beta.-trifluoroalanine (Faraci, W. S.; Walsh, C. T., "Mechanism of Inactivation of Alanine Racemase by .beta.,.beta.,.beta.-Trifluoroalanine." Biochemistry 1989, 28:431-437). ##STR9##
b. Therapeutic Potential
From a medicinal point of view, several enzymes in this class are very important targets for the development of specific inhibitors. DOPA decarboxylase is the target of the important commercial drug .alpha.-methyl-DOPA used to treat hypertension. Peripheral DOPA DC inhibitors are used in combination therapy with L-DOPA for the treatment of Parkinsonism. Inhibitors of lysine DC are potential anti-mycoplasmic agents. As arginine DC is found in bacterial, but not in mammalian systems, arginine DC inhibitors are potential antibiotics. Inhibitors of histidine DC, ornithine DC and SAM DC all have potential as antitumor agents. In particular, the latter two enzymes control flux through the polyamine pathway. They are induced in response to various trophic influences and are essential for rapid cell proliferation. Indeed, inhibitors of these enzymes have proven to be potent antiproliferative agents in tissue culture. A SAM DC inhibitor cures African sleeping sickness in mice and .alpha.-difluoromethylomithine is effective against the microorganism that produces pneumonia in AIDS patients. However, the effectiveness of these compounds as drugs is often compromised by compensatory mechanisms triggered by tumor cells [i.e. ornithine DC gene amplification or replacement of putrescine (ornithine DC product) with cadaverine (lysine DC product)]. This has stimulated the development of SAM DC inhibitors as potential antineoplastics, to be used in combination with ornithine DC inactivators. But most importantly, the reality of such tumor cell compensatory mechanisms underlines the need to develop DC inhibitors of fundamentally new structural classes.